1. Field of Invention
The present invention relates to a substrate for detecting helicase activity in a herpes simplex virus helicase as well as methods for using the substrates.
2. Description of Related Art
Helicases are a class of enzymes that catalyze unwinding of duplex DNA by disrupting the hydrogen bonds that hold the two strands of duplex DNA together. Matson and Kaiser-Rogers, Annu. Rev. Biochem. 59: 289-329 (1994); Lohman and Bjornson, Annu. Rev. Biochem. 65: 169-214 (1996). This unwinding process is important to generate single stranded DNA regions, which are subsequently utilized by other enzymes during DNA replication, repair, and recombination. Kornberg and Baker, DNA Replication, 2.sup.nd ed., W. H. Freeman and Co., New York (1992). Although the detailed unwinding mechanisms of helicases are not fully understood, it is clear that in order to unwind duplex DNA continuously, a helicase enzyme translocates , along the DNA by hydrolyzing nucleotide 5'-triphosphate (NTP or dNTP). The helicases thus far identified also exhibit a specific directionality, i.e., 3'-5' or 5'-3', for unwinding duplex DNA.
Helicase activity can be measured as either displacement of single stranded DNA (ssDNA) from duplex DNA or as continuous unwinding of duplex DNA. The most common assay is a gel-based helicase assay using a partially duplex M13 or .PHI.X174 DNA substrate. The ssDNA or ssRNA product, which is radiolabeled, can be detected by its altered mobility during electrophoresis on a nondenaturing gel, or by sensitivity to ssDNA-specific nucleases. Abdel-Monem et al., Eur. J. Biochem. 65, 441-449 (1976); Doguet et al., Cold Spring Harbor Symp. Quant. Biol. 43, 335-343 (1979); Kuhn et al., Cold Spring Harbor Symp. Quant. Biol. 43, 63-67 (1979); Palas et al., J. Biol. Chem. 265, 3447-3454 (1990); Venkatesan et al., J. Biol. Chem. 257, 12426-12434 (1982); Matson et al., J. Biol. Chem. 258,14017-14024 (1983). Direct measurement by electron microscopy has also been employed to visualize the regions of DNA unwound by helicases. Taylor and Smith, Cell, 22, 447-457 (1980); Baumel et al., Eur. J. Biochem. 138, 247-251 (1984); Benz et al., Acta Microbiologica Polonica 35, 191-197 (1986); Dodson et al., Science, 238, 964-967 (1987); Runyon et al., Proc. Natl. Acad. Sci. USA, 87, 6383-6387(1990).
Several continuous fluorometric assays have also been developed for measuring helicase activity. One assay requires a single-strand DNA binding (SSB) protein as the reporter molecule. Roman and Kowalczykowski, Biochemistry, 28, 2863-2873 (1989). As the duplex DNA is unwound, the SSB protein binds to the ssDNA products, and its intrinsic fluorescent emission is quenched. Another assay utilizes duplex DNA whose complementary ends are tagged with different fluorescent moieties. Houston and Kodadek, Proc. Natl. Acad. Sci. USA, 91, 5471-5474 (1994); Bjornson et al., Biochemistry, 33, 14306-14316 (1994). A change in the overall fluorescent emission is detected and quantitated as the duplex DNA is unwound. Similarly, DNA substrates can be modified with 2-aminopurine substituted in place of adenine. Raney et al., Proc. Natl. Acad. Sci. USA, 91, 6644-6648 (1994). The 2-aminopurine exhibits fluorescence quenching when the analog forms hydrogen bonds with thymine residues. Single stranded DNA products generated by a helicase will remove this quenching effect, resulting in an increase in fluorescent emission. Recently, different fluorophores that bind to duplex DNA have also been evaluated as reporter molecules during unwinding Eggleston et al., Nucleic acids Res. 24, 1179-1186 (1996). In this assay, the fluorescent reporter dye initially bound to the substrate is converted to free molecules in solution as the helicase unwinds the substrate, resulting in a reduction of the fluorescence signal.
Although the assays described above have been widely employed for purifying or characterizing several different types of helicases, these assays lack the ability to selectively test for a particular protein exhibiting helicase activity. A need thus exists for an assay which is specific for a particular type or class of helicase.
Herpes viruses are significant infectious agents which cause a variety of diseases in humans and other mammals. Herpes simplex viruses (HSVs) were the first of the human herpes viruses to be discovered and have been thoroughly characterized. The replication of HSV-1 DNA serves as an excellent model system to understand DNA replication by other herpes viruses. The herpes simplex virus type 1 (HSV-1) genome consists of 152 kb of linear dsDNA and contains three highly homologous origins of replication; ori.sub.L and two copies of ori.sub.S. Stow, EMBO J. 1, 863-867 (1982); Stow, Virology, 130, 427-438 (1983); Stow, J. Gen. Virol. 66, 3142; Weller et al., Mol. Cell. Biol. 5, 930-942 (1988); Challberg and Kelly, Annu. Rev. Biochem. 58, 671-717. The virus also encodes a 94-kDa origin binding protein, the product of the UL9 gene, which has been shown to be essential for replication of HSV-1 DNA in vivo. Elias et al., Proc. Natl. Acad. Sci. USA 83, 6322-6326 (1986); Olivo et al., Proc. Natl. Acad. Sci. USA 85, 5414-5418 (1988); Wu et al., J. Virol. 62, 435-443 (1988); Stow, J. Gen. Virol. 73, 313-321 (1992). The UL9 protein has also been shown to specifically and cooperatively bind the two inverted pentanucleotide repeats in Boxes I and II of ori.sub.S which are separated from each other by an A/T-rich sequence of 18 nucleotides (nt), illustrated in FIG. 2A. Elias et al., J. Biol. Chem. 265,17167-17173; Bruckner et al., J. Biol. Chem. 266, 2669-2674 (1991); Fierer and Challberg, J. Virol. 66, 39868-3995; Hazuda et al., J. Biol. Chem. 265,14309-14315 (1992). Another herpes viral origin, ori.sub.L contains a second copy of Box I in place of Box II, also illustrated in FIG. 2A.
In addition to its origin binding activity, the UL9 protein has been shown to exhibit DNA-dependent ATPase activity and 3'-5' helicase activity using a partially duplex M13 DNA substrate. Bruckner et al., J. Biol. Chem. 266, 2669-2674 (1991); Fierer and Challberg, J. Virol. 66, 39868-3995; Dodson and Lehman, J. Biol. Chem. 268,1213-1219 (1993); Boehmer et al., J. Biol. Chem. 268,1220-1225 (1993).
Despite the UL9 protein's intrinsic 3'-5' helicase activity on the partially duplex M13 DNA substrate, attempts to get the UL9 protein to unwind the viral origin sequence or the duplex Box I element have been unsuccessful. Attempts to demonstrate the specific unwinding of ori.sub.S by the UL9 protein were patterned after similar studies with the simian virus 40 large T antigen. In this system, a double hexamer protein binds, and by virtue of its helicase activity, unwinds the SV40 origin of replication. Stillman, Annu. Rev. Cell Biol. 5, 915-918 (1989); Boroweic et al., Cell 60,181-184 (1990). In vitro, a single stranded binding protein (SSB), such as either the homologous RP-A or the heterologous E. coli SSB, is required to maintain the separated strands. However, similar experiments with the herpes UL9 protein using both linear duplexes containing the ori.sub.S sequence or plasmids into which the ori.sub.S sequence had been inserted failed to demonstrate specific unwinding by the UL9 protein either alone or in the presence of the herpes SSB, ICP8, or heterologous SSBs. A need thus also exists for a method for detecting helicase activity in the UL9 protein when bound to a herpes origin of replication.
The HSV-1 single strand DNA binding protein, ICP8, was one of the first HSV-1 DNA replication proteins to be identified and has been shown to be required for the replication of HSV-1 DNA in vivo. Bayliss et al., Virology, 68, 124-134 (1975); Quinn and McGeoch, Nucleic Acids Res. 13, 8143-8163. Biochemical studies have shown that ICP8 binds single-stranded DNA rapidly and cooperatively and with at least fivefold greater affinity than double-stranded DNA. Ruyechan, J. Virol. 46, 661-666 (1983); Lee and Knipe, J. Virol. 54, 731-738 (1985). The DNA binding site size for ICP8 has been estimated from nuclease protection and electron microscopy to be 12-22 nucleotides. Boehmer and Lehman, J. Virol. 67, 711-715 (1993); Dutch and Lehman, J. Virol. 67, 6945-6949 (1993); Hernandez and Lehman, J. Biol. Chem. 265,11227-11232 (1990); O'Donnell et al., J. Biol. Chem. 262, 4260-4266 (1987). ICP8 is believed to play a key role in the assembly of the HSV-1 DNA replication proteins into prereplicative sites that are precursors to discrete nuclear locations in which viral DNA replication occurs. Quinlan et al., Cell, 36, 857-868 (1984); Gao and Knipe, J. Virol. 63, 5258-5267 (1989); Olivo et al., J. Virol. 63,196-204 (1989); de Bruyn Kops and Knipe, Cell, 55, 857-868 (1988). Further elucidation of the role ICP8 plays in the replication of HSV-1 is needed.